SIRE1 RETROTRANSPOSONS IN BARLEY (Hordeum vulgare L.).

Sireviruses are genera of copia LTR retrotransposons with a unique genome structure among retrotransposons. Barley (Hordeum vulgare L.) is an economically important plant. In this study, we used mature barley embryos, 10-day-old roots and 10-day-old leaves derived from the same barley plant to investigate SIRE) retrotransposon movements by Inter-Retrotransposon Amplified Polymorphism (IRAP) technique. We found polymorphism rates between 0-64% among embryos, roots and leaves. Polymorphism rates were detected to be 0-27% among embryos, 8-60% among roots, and 11-50% among leaves. Polymorphisms were observed not only among the parts of different individuals, but also on the parts of the same plant (23-64%). The internal domains of SIRE1 (GAG, ENV and RT) were also analyzed in the embryos, roots and leaves. Analysis of band profiles showed no polymorphism for GAG, however, different band patterns were observed among samples for RT and ENV. The sequencing of SIRE1 GAG, ENV and RT domains revealed 79% similarity for GAG, 96% for ENV and 83% for RT to copia retrotransposons. Comparison between barley retrotransposons and SIRE1 in barley indicated that SIRE1-GAG, ENV and RT might be diverge earlier from barley retrotransposons. SIRE1 sequences were compared with SIRE1 in barley, results showed the closest homologues were SIRE1-ENVand SIRE1-RTsequences, and SIRE1-GAG sequences was a sister group to sequences of Glycine max. This study is the first detailed investigation of SIRE1 in barley genome. The obtained findings are expected to contribute to the comprehension of SIRE1 retrotransposon and its role in barley genome.

CYTOTOXIC EFFECTS OF METAPHASE-ARRESTING METHODS IN BARLEY.

Metaphase-arresting agents amiprophos-methyl (APM), colchicine (COL) and cell cycle-synchronization (CCS) with APM and hydroxyurea (HU) were tested for growth, metaphase index and cytogenetic abnomalities in barley (Hordeum vulgare cv. Bornova-92). Seeds were germinated for 2 days and then seedlings were treated with 8 µM (2.4 mg/l) APM for 2 h or 1.25 mM (0.5 g/l) COL or synchronized (CCS) with 1.25 mM (95 mg/l) hydroxyurea for 18 h and with 4 µM (1.2 mg/l) APM for 2 h. APM and CCS caused metaphase indices 12.57 and 38.82% respectively. COL also arrested metaphase (14.10%) but also resulted in nuclear aberrations (11.15%). After removal of APM and CCS, cells were released to grow and divide. However, COL caused irreversible effects on cell division and growth and meanwhile was shown to be effective for micronucleus formation.

Variations in BARE-1 insertion patterns in barley callus cultures.

The stability of aging barley calli was investigated with the barley retroelement 1 (BARE-1) retrotransposon specific inter-retrotransposon amplified polymorphism (IRAP) technique. Mature embryos of barley (Hordeum vulgare cv. Zafer-160) were cultured on callus induction MS medium supplemented with 3 mg/L 2,4-D and maintained on the same medium for 60 days. Ten IRAP primers were used in 25 different combinations. The similarity index between 30-day-old and 45-day-old calli was 84%; however, the similarity index between mature embryos and 45-day-old calli was 75%. These culture conditions caused BARE-1 retrotransposon alterations to appear as different band profiles. This is the first report of the use of the IRAP technique in barley in an investigation of callus development.

Effects of hyperbaric oxygenation on cultured barley embryos.

Changes in relative water content (RWC), lipid peroxidation, proline and antioxidant system in relation to the tolerance to oxidative stress enzymes mediated high pressure were investigated in Hordeum vulgare L. cv. Tokak. For this purpose, mature embryos cultured on MS media were treated in a hyperbaric oxygenation chamber (approx. 59.06 feets, 2 kp/cm2) with pure oxygen for 60 minutes/day for a growth period of ten days in a plant growth chamber. Constitutive activities of SOD, APOX, GR and POX were higher in hyperbaric oxygenated (HBO) explants, being 96.07%, 28.57%, 77.77% and 54.14% for the 5th days; 95.78%, 40%, 37.5%, and 94.98% for the 10th days of culture, respectively, than in the control plants. Increase in SOD activity was also shown on polyacrilamide gel electrophoresis on the 10th day of application. Proline accumulation was increased in HBO-treated explants both on the 5th days (85.71%) and 10th days (37.14%) of treatment. MDA content was found to be higher in HBO treated explants both on the 5th (53.84%) and 10th (59.83%) days of culture.

Antibacterial activity of seed proteins of Robinia pseudoacacia.

A low molecular weight cationic peptide was isolated from Robinia pseudoacacia seed and tested in vitro against seven bacteria (Corynebacterium michiganense, Staphylococcus aureus, Bacillus subtilis, Erwinia carotovora subsp. carotovora, Pseudomonas syringae pv syringae, Xanthomonas campestris pv campestris, and Escherichia coli). The peptide inhibited the growth of the tested strains. The effective concentrations required for 50% inhibition of bacterial growth ranged between 20 and 120 microg ml(-1) protein. S. aureus was found to be the most sensitive strain, however, E. coli was not affected much when compared with others. Reduction of antibacterial activity of the peptide with CaCl2 addition into the growth medium was also observed.

Optimization of gene transfer into barley (Hordeum vulgare L.) mature embryos by tissue electroporation.

This study was conducted to detect the optimum conditions for DNA transfer into mature embryos of barley via electroporation. Cultured mature embryos of barley were directly electroporated in the presence of the pBI 121 vector carrying both the ß-glucuronidase (GUS) and neomycin phosphotransferase II (npt II) genes. It was found that 500 v/cm and 500 µFd capacitance was the optimum combination for healthy germination of the transformed plants from mature electroporated embryos. Effects of culture duration before electroporation and selection antibiotic concentrations on germination were also examined. Gene transfer performed on 3-day-old cultures resulted in the highest germination frequencies. GUS expression was observed on transversal sections of embryos and mature leaves from 3 month-old regenerants. PCR and Southern blot analyses show the presence of the npt II transgene in the genome of a plant.

Direct somatic embryogenesis and synthetic seed production from Paulownia elongata.

We have developed a reproducible system for efficient direct somatic embryogenesis from leaf and internodal explants of Paulownia elongata. The somatic embryos obtained were subsequently encapsulated as single embryos to produce synthetic seeds. Several plant growth regulators [6-benzylaminopurine, indole-3-acetic acid, alpha-naphthaleneacetic acid, kinetin and thidiazuron (TDZ)] alone or in combination were tested for their capacity to induce somatic embryogenesis. The highest induction frequencies of somatic embryos were obtained on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% Phytagel, 500 mg l(-1) casein hydrolysate and 10 mg l(-1) TDZ (medium MS10). Somatic embryos were induced from leaf (69.8%) and internode (58.5%) explants on MS10 medium after 7 days. Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal media. The maturation frequency of somatic embryos from leaf and internodal explants was 50.8% and 45.8%, respectively. Subculturing of mature embryos led to their germination on the same medium with a germination frequency of 50.1% and 29.8% from leaf and internode explants, respectively. Somatic embryos obtained directly on leaf explants were used for encapsulation in liquid MS medium containing different concentrations of sodium alginate with a 30-min exposure to 50 m M CaCl(2). A 3% sodium alginate concentration provided a uniform encapsulation of the embryos with survival and germination frequencies of 73.7% and 53.3%, respectively. Storage at 4 degrees C for 30 days or 60 days significantly reduced the survival and complete germination frequencies of both encapsulated and non-encapsulated embryos relative to those of non-stored somatic embryos. However, the survival and germination rates of encapsulated embryos increased following storage at 4 degrees C. After 30 days or 60 days of storage, the survival rates of encapsulated embryos were 67.8% and 53.5% and the germination frequencies were 43.2% and 32.4%, respectively. These systems could be useful for the rapid clonal propagation and dissemination of synthetic seed material of Paulownia elongata.

AFLP marker linked to water-stress-tolerant bulks in barley (Hordeum vulgare L)

The amplified fragment length polymorphism (AFLP) assay is an efficient method for the identification of molecular markers, useful in the improvement of numerous crop species. Bulked Segregant Analysis (BSA) was used to identify AFLP markers associated with water-stress tolerance in barley, as this would permit rapid selection of water-stress tolerant genotypes in breeding programs. AFLP markers linked to water-stress tolerance was identified in two DNA pools (tolerant and sensitive), which were established using selected F-2 individuals resulting from a cross between water-stress-tolerant and sensitive barley parental genotypes, based on their paraquat (PQ) tolerance, leaf size, and relative water content (RWC). All these three traits were previously shown to be associated with water-stress tolerance in segregating F-2 progeny of the barley cross used in a previous study. AFLP analysis was then performed on these DNA pools, using 40 primer pairs to detect AFLP fragments that are present/absent, respectively, in the two pools and their parental lines. One separate AFLP fragment, which was present in the tolerant parent and in the tolerant bulk, but absent in the sensitive parent and in the sensitive bulk, was identified. Polymorphism of the AFLP marker was tested among tolerant and sensitive F-2 individuals. The presence of this marker that is associated with water-stress tolerance will greatly enhance selection for paraquat and water-stress tolerant genotypes in future breeding programs